Crystallization and structure determination of glycosylated human beta secretase, an enzyme implicated in alzheimer&#39;s disease

ABSTRACT

An inhibitor bound form of human beta secretase, also known as memapsin 2 and BACE, particularly in a glycosylated form as expressed in Chinese hamster ovary (CHO), HEK293 cells, or in insect cells as part of a Baculovirus expression system has been crystallized, and the three dimensional x-ray crystal structure has been solved to 3.2 Å resolution. The x-ray crystal structure is useful for solving the structure of other molecules or molecular complexes, and designing inhibitors of human beta secretase activity.

RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 10/027,277, filedDec. 21, 2001 (abandoned), which is a continuation of U.S. applicationSer. No. 09/808,262, filed Mar. 14, 2001 (abandoned), which is acontinuation-in-part of U.S. application Ser. No. 09/747,420, filed 23Dec. 2000 (abandoned), all of which are incorporated herein by referencein their entireties.

FIELD OF THE INVENTION

This invention relates to the crystallization and structuredetermination of beta secretase, also known as memapsin 2 and BACE, fromhuman (Homo sapiens), particularly in a glycosylated form as expressedin Chinese hamster ovary (CHO), HEK293 cells, or in insect cells as partof a Baculovirus expression system.

BACKGROUND

Alzheimer's disease (AD) causes progressive dementia with consequentformation of amyloid plaques, neurofibrillary tangles, gliosis andneuronal loss. The disease occurs in both genetic and sporadic formswhose clinical course and pathological features are quite similar. Threegenes have been discovered to date which, when mutated, cause anautosomal dominant form of Alzheimer's disease. These encode the amyloidprotein precursor (APP) and two related proteins, presenilin-1 (PS1) andpresenilin-2 (PS2), which, as their names suggest, are structurally andfunctionally related. Mutations in any of the three proteins have beenobserved to enhance proteolytic processing of APP via an intracellularpathway that produces amyloid beta peptide (Aβ peptide, or sometimeshere as Abeta), a 40-42 amino acid long peptide that is the primarycomponent of amyloid plaque in AD.

Dysregulation of intracellular pathways for proteolytic processing maybe central to the pathophysiology of AD. In the case of plaqueformation, mutations in APP, PS1 or PS2 consistently alter theproteolytic processing of APP so as to enhance formation of Aβ 1-42, aform of the Aβ peptide which seems to be particularly amyloidogenic, andthus very important in AD. Different forms of APP range in size from695-770 amino acids, localize to the cell surface, and have a singleC-terminal transmembrane domain. The Abeta peptide is derived from aregion of APP adjacent to and containing a portion of the transmembranedomain. Normally, processing of APP at the α-secretase site cleaves themidregion of the Aβ sequence adjacent to the membrane and releases thesoluble, extracellular domain of APP from the cell surface. Thisα-secretase APP processing creates soluble APP-α, which is normal andnot thought to contribute to AD. Pathological processing of APP at theβ- and γ-secretase sites, which are located N-terminal and C-terminal tothe α-secretase site, respectively, produces a very different resultthan processing at the α site. Sequential processing at the β- andγ-secretase sites releases the Aβ peptide, a peptide possibly veryimportant in AD pathogenesis. Processing at the β- and γ-secretase sitescan occur in both the endoplasmic reticulum (in neurons) and in theendosomal/lysosomal pathway after reinternalization of cell surface APP(in all cells). Despite intense efforts, for 10 years or more, toidentify the enzymes responsible for processing APP at the β and γsites, to produce the Aβ peptide, those proteases remained unknown untilrecently.

The identification and characterization of the β secretase enzyme,termed Aspartyl Protease 2 (Asp2) has been established. In addition, theX-ray crystal structure of human beta secretase in complex with apeptide inhibitor was solved and published Hong et al., Science 290:150-53 (2000) from protein expressed in E. coli that contained nocovalent sugar (glycosylation) at any of the four putative glycosylationsites within the enzyme.

SUMMARY OF THE INVENTION

This invention relates to the crystallization and structuredetermination of beta secretase, also known as memapsin 2 and BACE, fromhuman (Homo sapiens), particularly in a glycosylated form as expressedin Chinese hamster ovary (CHO), HEK293 cells, or in insect cells as partof a Baculovirus expression system.

In one aspect, the present invention provides a method for crystallizinga human beta secretase molecule or molecular complex. The methodinvolves crystallizing a human beta secretase molecule or molecularcomplex by preparing purified human beta secretase in the presence of aninhibitor and crystallizing human beta secretase from a solution havinga pH of about 3.5 to about 5.5.

In another aspect, the present invention provides crystalline forms of ahuman beta secretase molecule. In one embodiment, the present inventionprovides a crystal of beta secretase having the trigonal space groupsymmetry P3₂21. In another embodiment, a crystal of human beta secretaseis provided having the trigonal space group symmetry P3₂21 with unitcell dimensions of a, b, and c, wherein a is about 77 Å to about 147 Å,b is about 77 Å to about 147 Å, and c is about 77 Å to about 147 Å; andα=β=90°, and γ=120°. Preferably, the crystal has unit cell dimensions ofa=112.0 Å, b=112 Å, c=110 Å, α=β=90 °, γ=120°.

In another aspect, the present invention provides a method of producinghuman beta secretase, the method including expressing the human betasecretase in a mammalian cell line.

In another aspect, the present invention provides a method of producinghuman beta secretase, the method including expressing the human betasecretase in an insect cell line.

Abbreviations

The following abbreviations are used throughout this disclosure:

Alzheimer's disease (AD)

Amyloid beta peptide (Aβ peptide or Abeta)

Amyloid protein precursor (APP)

Aspartyl protease 2 (Asp2)

Baculovirus expression system (BVES)

Beta secretase (memapsin 2, BACE)

Chinese hamster ovary (CHO)

Dimethyl sulfoxide (DMSO)

Multiple anomalous dispersion (MAD)

Presenilin-1 (PS1)

Presenilin-2 (PS2)

Polyethylene glycol (PEG)

The following amino acid abbreviations are used throughout thisdisclosure:

A = Ala = Alanine T = Thr = Threonine V = Val = Valine C = Cys =Cysteine L = Leu = Leucine Y = Tyr = Tyrosine I = Ile = Isoleucine N =Asn = Asparagine P = Pro = Proline Q = Gln = Glutamine F = Phe =Phenylalanine D = Asp = Aspartic Acid W = Trp = Tryptophan E = Glu =Glutamic Acid M = Met = Methionine K = Lys = Lysine G = Gly = Glycine R= Mg = Arginine S = Ser = Serine H = His = Histidine

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of the chemical structure of an inhibitor usedin co-crystallization experiments,N1-((2S,3R)-4-(3-iodobenzylamino)-1-(3,5-difluorophenyl)-3-hydroxybutan-2-yl)-5-methyl-N3,N3-dipropylbenzene-1,3-diamide.

FIG. 2 is the synthetic peptideSer-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Arg-Gly-Gly-Cys (where Sta=statine)(SEQ ID NO:3) PNU-292593E used for affinity purification of betasecretase.

FIG. 3 is an electron density map for the inhibitor illustrated in FIG.1 at 3.2 Å.

FIG. 4 is a stereo view of a C^(α) trace of human beta secretase (blackline) in the presence of the inhibitor illustrated in FIG. 1. Thegeneral location of the inhibitor trace (gray line) is indicated by anarrow.

FIG. 5 is a stereo view of C^(α) traces of human beta secretase as asuperposition of a data set from the present invention (black line) witha data set from Hong et al., Science 290:150-53 (2000) (gray line). Ther.m.s. deviation on C^(α) between the illustrated data sets was 0.43 Å.

FIG. 6 depicts a stereo view of the active site of human beta secretase(illustrated with light gray carbons, dark gray oxygens, and blacknitrogens) with the inhibitor illustrated in FIG. 1. The generallocation of the inhibitor is indicated by an arrow (illustrated withlight gray carbons, dark gray oxygens, and black nitrogens). The iodineatom is circled and the two fluorine atoms are indicated by a “•”symbol.

FIG. 7 depicts the sequence (SEQ ID NO:1) of residues for recombinanthuman beta secretase present in the X-ray structure.

DETAILED DESCRIPTION OF THE INVENTION Crystalline Form(s) and Method ofMaking

The three-dimensional structure of human beta secretase was solved usingx-ray crystallography to 3.2 Å resolution. Accordingly, the inventionincludes a human beta secretase crystal and/or a crystal with human betasecretase co-crystallized with a ligand, such as an inhibitor.Preferably, the crystal has trigonal space group symmetry P3₂21. Morepreferably, the crystal includes hexagonal shaped unit cells, each unitcell having dimensions a=112.0±35 Å, b=112±35 Å, c=110±35 Å, α=β=90°,γ=120. The crystallized enzyme is a monomer with a single monomer in theasymmetric unit.

According to the present invention, human beta secretase can be isolatedfrom a variety of cell lines, for example, the mammalian cell lineCHO-K1 or an insect cell line.

In a preferred embodiment, molecular complexes of purified human betasecretase at a concentration of about 1 mg/ml to about 80 mg/ml may becrystallized in the presence of an inhibitor at a concentration fromabout 0.1 to about 10 mM, for example, by using a streak seedingprocedure from a solution including about 5% by weight to about 50% byweight PEG or PEG-MME (PEG monomethyl ether), PEG-DME (PEG dimethylether), or polyoxyalkylenepolyamines (e.g., materials available underthe trade designation JEFFAMINE from Huntsman Corp., Salt Lake City,Utah) (preferably having a number average molecular weight between about200 and about 20,000), preferably, a salt (more preferably about 0.001 Mto about 0.5 M salt), and about 0% by weight to about 20% by weightorganic solvent (such as DMSO), wherein the solution is buffered to a pHof about 3.5 to about 5.5 (preferably, a pH of about 4.0 to about 4.7).Exemplary salts include sodium chloride, ammonium sulfate, magnesiumsulfate, lithium sulfate, or combinations thereof. Use of a bufferhaving a pK_(a) of about 3 to about 6 is preferred. A “molecularcomplex” means a protein in covalent or non-covalent association with achemical entity. A buffer having a pK_(a) of between about 3 and 6 ispreferred for use in the crystallization method. A particularlypreferred buffer is about 10 mM to about 200 mM sodium acetate.Variation in buffer and buffer pH as well as other additives such as PEGor PEG-MME (PEG monomethyl ether), PEG-DME (PEG dimethyl ether), orpolyoxyalkylenepolyanines (e.g., materials available under the tradedesignation JEFFAMINE from Huntsman Corp., Salt Lake City, Utah) isapparent to those skilled in the art and may result in similar crystals.

The invention further includes an human beta secretase crystal that isisomorphous with an human beta secretase crystal characterized by a unitcell having dimensions of a, b, and c; wherein a is about 77 Å to about147 Å, b is about 77 Å to about 147 Å, and c is about 75 Å to about 145Å; and α=β=90°, and γ=120°.

X-Ray Crystallographic Analysis

Each of the constituent amino acids of human beta secretase is definedby a set of structure coordinates. The term “structure coordinates”refers to Cartesian coordinates derived from mathematical equationsrelated to the patterns obtained on diffraction of a monochromatic beamof x-rays by the atoms (scattering centers) of an human beta secretasecomplex in crystal form. The diffraction data are used to calculate anelectron density map of the repeating unit of the crystal. The electrondensity maps are then used to establish the positions of the individualatoms of the human beta secretase protein or protein/ligand complex.

Slight variations in structure coordinates can be generated bymathematically manipulating the human beta secretase or human betasecretase/ligand structure coordinates. For example, structurecoordinates could be manipulated by crystallographic permutations of thestructure coordinates, fractionalization of the structure coordinates,integer additions or subtractions to sets of the structure coordinates,inversion of the structure coordinates or any combination of the above.Alternatively, modifications in the crystal structure due to mutations,additions, substitutions, and/or deletions of amino acids, or otherchanges in any of the components that make up the crystal, could alsoyield variations in structure coordinates. Such slight variations in theindividual coordinates will have little effect on overall shape. If suchvariations are within an acceptable standard error as compared to theoriginal coordinates, the resulting three-dimensional shape isconsidered to be structurally equivalent. Structural equivalence isdescribed in more detail below.

It should be noted that slight variations in individual structurecoordinates of the human beta secretase would not be expected tosignificantly alter the nature of chemical entities such as ligands thatcould associate with the inhibitor binding pockets. In this context, thephrase “associating with” refers to a condition of proximity between achemical entity, or portions thereof, and an human beta secretasemolecule or portions thereof. The association may be non-covalent,wherein the juxtaposition is energetically favored by hydrogen bonding,van der Waals forces, or electrostatic interactions, or it may becovalent.

Thus, for example, a ligand that bound to a inhibitor binding pocket ofhuman beta secretase would also be expected to bind to or interfere withanother inhibitor binding pocket whose structure coordinates define ashape that falls within the acceptable error.

It will be readily apparent to those of skill in the art that thenumbering of amino acids in other isoforms of human beta secretase maybe different than that of human beta secretase expressed in CHO or HKE293 cells.

Active Site and Other Structural Features

Applicants' invention provides information about the shape and structureof the inhibitor binding pocket of human beta secretase in the presenceof an inhibitor. The secondary structure of the human beta secretasemonomer includes two domains consistent with a typical aspartic proteasefold.

Binding pockets are of significant utility in fields such as drugdiscovery. The association of natural ligands or substrates with thebinding pockets of their corresponding receptors or enzymes is the basisof many biological mechanisms of action. Similarly, many drugs exerttheir biological effects through association with the binding pockets ofreceptors and enzymes. Such associations may occur with all or any partsof the binding pocket. An understanding of such associations helps leadto the design of drugs having more favorable associations with theirtarget, and thus improved biological effects. Therefore, thisinformation is valuable in designing potential inhibitors of betasecretase-like inhibitor binding pockets, as discussed in more detailbelow.

The term “binding pocket,” as used herein, refers to a region of amolecule or molecular complex, that, as a result of its shape, favorablyassociates with another chemical entity. Thus, a binding pocket mayinclude or consist of features such as cavities, surfaces, or interfacesbetween domains. Chemical entities that may associate with a bindingpocket include, but are not limited to, cofactors, substrates,inhibitors, agonists, and antagonists.

The amino acid constituents of an human beta secretase inhibitor bindingpocket as defined herein are positioned in three dimensions. In oneaspect, the structure coordinates defining a inhibitor binding pocket ofhuman beta secretase include structure coordinates of all atoms in theconstituent amino acids; in another aspect, the structure coordinates ofa inhibitor binding pocket include structure coordinates of just thebackbone atoms of the constituent atoms.

The inhibitor binding pocket of human beta secretase preferably includesthe amino acids listed in Table 1, more preferably the amino acidslisted in Table 2, and most preferably the amino acids listed in Table3. Alternatively, the inhibitor binding pocket of human beta secretasemay be defined by those amino acids whose backbone atoms are situatedwithin about 4 Å, more preferably within about 7 Å, most preferablywithin about 10 Å, of one or more constituent atoms of a bound substrateor inhibitor. In yet another alternative, the inhibitor binding pocketmay be defined by those amino acids whose backbone atoms are situatedwithin a sphere centered on the coordinates representing the alphacarbon atom of residue Thr 231, the sphere having a radius of about 15Å, preferably about 20 Å, and more preferably about 25 Å.

The term “beta secretase-like inhibitor binding pocket” refers to aportion of a molecule or molecular complex whose shape is sufficientlysimilar to at least a portion of a inhibitor binding pocket of humanbeta secretase as to be expected to bind related structural analogues. Astructurally equivalent inhibitor binding pocket is defined by a rootmean square deviation from the structure coordinates of the backboneatoms of the amino acids that make up inhibitor binding pockets in humanbeta secretase of at most about 0.35 Å. How this calculation is obtainedis described below.

Accordingly, the invention provides molecules or molecular complexesincluding an human beta secretase inhibitor binding pocket or betasecretase-like inhibitor binding pocket, as defined by the sets ofstructure coordinates described above.

TABLE 1 Residues with 4 Å of inhibitor binding site. GLY 11 SER 35 PHE108 ASP 228 GLY 13 PRO 70 ILE 110 GLY 230 LEU 30 TYR 71 TRP 115 THR 231ASP 32 THR 72 ILE 126 THR 232 GLY 34 GLN 73 TYR 198 ARG 235

TABLE 2 Residues with 7 Å of inhibitor binding site. SER 10 ASN 37 PHE109 GLY 230 GLY 11 VAL 69 ILE 110 THR 231 GLN 12 PRO 70 TRP 115 THR 232GLY 13 TYR 71 ILE 118 ASN 233 TYR 14 THR 72 ILE 126 ARG 235 LEU 30 GLN73 ALA 127 SER 325 VAL 31 GLY 74 ARG 128 THR 329 ASP 32 LYS 75 TYR 198VAL 332 THR 33 TRP 76 LYS 224 ALA 335 GLY 34 ASP 106 ILE 226 SER 35 LYS107 ASP 228 SER 36 PHE 108 SER 229

TABLE 3 Residues with 10 Å of inhibitor binding site. ARG 7 TYR 71 LEU121 THR 232 GLY 8 THR 72 ALA 122 ASN 233 LYS 9 GLN 73 TYR 123 LEU 234SER 10 GLY 74 ALA 124 ARG 235 GLY 11 LYS 75 GLU 125 ARG 307 GLN 12 TRP76 ILE 126 PHE 322 GLY 13 GLU 77 ALA 127 ALA 323 TYR 14 ILE 102 ARG 128ILE 324 TYR 15 SER 105 PRO 129 SER 325 ILE 29 ASP 106 LEU 154 GLN 326LEU 30 LYS 107 TRP 197 SER 327 VAL 31 PHE 108 TYR 198 SER 328 ASP 32 PHE109 TYR 199 THR 329 THR 33 ILE 110 ASP 223 GLY 330 GLY 34 ASN 111 LYS224 THR 331 SER 35 SER 113 SER 225 VAL 332 SER 36 TRP 115 ILE 226 MET333 ASN 37 GLU 116 VAL 227 GLY 334 ALA 39 GLY 117 ASP 228 ALA 335 TYR 68ILE 118 SER 229 VAL 336 VAL 69 LEU 119 GLY 230 MET 338 PRO 70 GLY 120THR 231 GLU 339Three-Dimensional Configurations

X-ray structure coordinates define a unique configuration of points inspace. Those of skill in the art understand that a set of structurecoordinates for protein or an protein/ligand complex, or a portionthereof, define a relative set of points that, in turn, define aconfiguration in three dimensions. A similar or identical configurationcan be defined by an entirely different set of coordinates, provided thedistances and angles between coordinates remain essentially the same. Inaddition, a scalable configuration of points can be defined byincreasing or decreasing the distances between coordinates by a scalarfactor while keeping the angles essentially the same.

The present invention thus includes the scalable three-dimensionalconfiguration of points derived from the structure coordinates of atleast a portion of an human beta secretase molecule or molecularcomplex, as well as structurally equivalent configurations, as describedbelow. Preferably, the scalable three-dimensional configuration includespoints derived from structure coordinates representing the locations ofa plurality of the amino acids defining an human beta secretaseinhibitor binding pocket.

In one embodiment, the scalable three-dimensional configuration includespoints derived from structure coordinates representing the locations thebackbone atoms of a plurality of amino acids defining the human betasecretase inhibitor binding pocket, preferably the amino acids listed inTable 1, more preferably the amino acids listed in Table 2, and mostpreferably the amino acids listed in Table 3. Alternatively, thescalable three-dimensional configuration includes points derived fromstructure coordinates representing the locations of the side chain andthe backbone atoms (other than hydrogens) of a plurality of the aminoacids defining the human beta secretase inhibitor binding pocket,preferably the amino acids listed in Table 1, more preferably the aminoacids listed in Table 2, and most preferably the amino acids listed inTable 3.

Likewise, the invention also includes the scalable three-dimensionalconfiguration of points derived from structure coordinates of moleculesor molecular complexes that are structurally homologous to betasecretase, as well as structurally equivalent configurations.Structurally homologous molecules or molecular complexes are definedbelow. Advantageously, structurally homologous molecules can beidentified using the structure coordinates of human beta secretaseaccording to a method of the invention.

The configurations of points in space derived from structure coordinatesaccording to the invention can be visualized as, for example, aholographic image, a stereodiagram, a model or a computer-displayedimage, and the invention thus includes such images, diagrams or models.

Structurally Equivalent Crystal Structures

Various computational analyses can be used to determine whether amolecule or a inhibitor binding pocket portion thereof is “structurallyequivalent,” defined in terms of its three-dimensional structure, to allor part of human beta secretase or its inhibitor binding pockets. Suchanalyses may be carried out in current software applications, such asthe Molecular Similarity application of QUANTA (Molecular SimulationsInc., San Diego, Calif.) version 4.1, and as described in theaccompanying User's Guide.

The Molecular Similarity application permits comparisons betweendifferent structures, different conformations of the same structure, anddifferent parts of the same structure. The procedure used in MolecularSimilarity to compare structures is divided into four steps: (1) loadthe structures to be compared; (2) define the atom equivalences in thesestructures; (3) perform a fitting operation; and (4) analyze theresults.

Each structure is identified by a name. One structure is identified asthe target (i.e., the fixed structure); all remaining structures areworking structures (i.e., moving structures). Since atom equivalencywithin QUANTA is defined by user input, for the purpose of thisinvention equivalent atoms are defined as protein backbone atoms (N,C^(α), C, and O) for all conserved residues between the two structuresbeing compared. A conserved residue is defined as a residue which isstructurally or functionally equivalent. Only rigid fitting operationsare considered.

When a rigid fitting method is used, the working structure is translatedand rotated to obtain an optimum fit with the target structure. Thefitting operation uses an algorithm that computes the optimumtranslation and rotation to be applied to the moving structure, suchthat the root mean square difference of the fit over the specified pairsof equivalent atom is an absolute minimum. This number, given inangstroms, is reported by QUANTA.

For the purpose of this invention, any molecule or molecular complex orinhibitor binding pocket thereof, or any portion thereof, that has aroot mean square deviation of conserved residue backbone atoms (N,C^(α), C, O) of less than about 0.35 Å, when superimposed on therelevant backbone atoms is considered “structurally equivalent” to thereference molecule. That is to say, the crystal structures of thoseportions of the two molecules are substantially identical, withinacceptable error. Particularly preferred structurally equivalentmolecules or molecular complexes are those that are defined by theentire set of structure coordinates ± a root mean square deviation fromthe conserved backbone atoms of those amino acids of not more than 0.35Å. More preferably, the root mean square deviation is less than about0.2 Å. Another embodiment of this invention is a molecular complex forthose amino acids listed in Table 1, ± a root mean square deviation fromthe conserved backbone atoms of those amino acids of not more than 0.35Å, preferably less than about 0.2 Å. Still another embodiment of thisinvention is a molecular complex for those amino acids listed in Table2, ± a root mean square deviation from the conserved backbone atoms ofthose amino acids of not more than 0.35 Å, preferably less than about0.2 Å.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations. It is a way to expressthe deviation or variation from a trend or object. For purposes of thisinvention, the “root mean square deviation” defines the variation in thebackbone of a protein from the backbone of human beta secretase or ainhibitor binding pocket portion thereof, as defined by the structurecoordinates of human beta secretase described herein.

Machine Readable Storage Media

Transformation of the structure coordinates for all or a portion ofhuman beta secretase or the human beta secretase/ligand complex or oneof its inhibitor binding pockets, for structurally homologous moleculesas defined below, or for the structural equivalents of any of thesemolecules or molecular complexes as defined above, intothree-dimensional graphical representations of the molecule or complexcan be conveniently achieved through the use of commercially-availablesoftware.

The invention thus further provides a machine-readable storage mediumincluding a data storage material encoded with machine readable datawhich, when using a machine programmed with instructions for using saiddata, is capable of displaying a graphical three-dimensionalrepresentation of any of the molecule or molecular complexes of thisinvention that have been described above. In a preferred embodiment, themachine-readable data storage medium includes a data storage materialencoded with machine readable data which, when using a machineprogrammed with instructions for using said data, is capable ofdisplaying a graphical three-dimensional representation of a molecule ormolecular complex including all or any parts of an human beta secretaseinhibitor binding pocket or an beta secretase-like inhibitor bindingpocket, as defined above. In another preferred embodiment, themachine-readable data storage medium includes a data storage materialencoded with machine readable data which, when using a machineprogrammed with instructions for using said data, is capable ofdisplaying a graphical three-dimensional representation of a molecule ormolecular complex ± a root mean square deviation from the backbone atomsof said amino acids of not more than 0.43 Å.

In an alternative embodiment, the machine-readable data storage mediumincludes a data storage material encoded with a first set of machinereadable data which includes the Fourier transform of structurecoordinates, and which, when using a machine programmed withinstructions for using said data, can be combined with a second set ofmachine readable data including the x-ray diffraction pattern of amolecule or molecular complex to determine at least a portion of thestructure coordinates corresponding to the second set of machinereadable data.

For example, a system for reading a data storage medium may include acomputer including a central processing unit (“CPU”), a working memorywhich may be, e.g., RAM (random access memory) or “core” memory, massstorage memory (such as one or more disk drives or CD-ROM drives), oneor more display devices (e.g., cathode-ray tube (“CRT”) displays, lightemitting diode (“LED”) displays, liquid crystal displays (“LCDs”),electroluminescent displays, vacuum fluorescent displays, field emissiondisplays (“FEDs”), plasma displays, projection panels, etc.), one ormore user input devices (e.g., keyboards, microphones, mice, trackballs, touch pads, etc.), one or more input lines, and one or moreoutput lines, all of which are interconnected by a conventionalbidirectional system bus. The system may be a stand-alone computer, ormay be networked (e.g., through local area networks, wide area networks,intranets, extranets, or the internet) to other systems (e.g.,computers, hosts, servers, etc.). The system may also include additionalcomputer controlled devices such as consumer electronics and appliances.

Input hardware may be coupled to the computer by input lines and may beimplemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems connected bya telephone line or dedicated data line. Alternatively or additionally,the input hardware may include CD-ROM drives or disk drives. Inconjunction with a display terminal, a keyboard may also be used as aninput device.

Output hardware may be coupled to the computer by output lines and maysimilarly be implemented by conventional devices. By way of example, theoutput hardware may include a display device for displaying a graphicalrepresentation of a binding pocket of this invention using a programsuch as QUANTA as described herein. Output hardware might also include aprinter, so that hard copy output may be produced, or a disk drive, tostore system output for later use.

In operation, a CPU coordinates the use of the various input and outputdevices, coordinates data accesses from mass storage devices, accessesto and from working memory, and determines the sequence of dataprocessing steps. A number of programs may be used to process themachine-readable data of this invention. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. References to components of the hardware system are included asappropriate throughout the following description of the data storagemedium.

Machine-readable storage devices useful in the present inventioninclude, but are not limited to, magnetic devices, electrical devices,optical devices, and combinations thereof. Examples of such data storagedevices include, but are not limited to, hard disk devices, CD devices,digital video disk devices, floppy disk devices, removable hard diskdevices, magneto-optic disk devices, magnetic tape devices, flash memorydevices, bubble memory devices, holographic storage devices, and anyother mass storage peripheral device. It should be understood that thesestorage devices include necessary hardware (e.g., drives, controllers,power supplies, etc.) as well as any necessary media (e.g., disks, flashcards, etc.) to enable the storage of data.

Structurally Homologous Molecules, Molecular Complexes, and CrystalStructures

Structure coordinates can be used to aid in obtaining structuralinformation about another crystallized molecule or molecular complex.The method of the invention allows determination of at least a portionof the three-dimensional structure of molecules or molecular complexeswhich contain one or more structural features that are similar tostructural features of human beta secretase. These molecules arereferred to herein as “structurally homologous” to human beta secretase.Similar structural features can include, for example, regions of aminoacid identity, conserved active site or binding site motifs, andsimilarly arranged secondary structural elements (e.g., α helices and βsheets). Optionally, structural homology is determined by aligning theresidues of the two amino acid sequences to optimize the number ofidentical amino acids along the lengths of their sequences; gaps ineither or both sequences are permitted in making the alignment in orderto optimize the number of identical amino acids, although the aminoacids in each sequence must nonetheless remain in their proper order.Preferably, two amino acid sequences are compared using the Blastpprogram, version 2.0.9, of the BLAST 2 search algorithm, as described byTatusova et al., FEMS Microbiol Lett 174, 247-50 (1999), and availableon the world wide web at ncbi.nlm.nih.gov/gorf/bl2.html. Preferably, thedefault values for all BLAST 2 search parameters are used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and filter on. In the comparison oftwo amino acid sequences using the BLAST search algorithm, structuralsimilarity is referred to as “identity.” Preferably, a structurallyhomologous molecule is a protein that has an amino acid sequence sharingat least 65% identity with a native or recombinant amino acid sequenceof human beta secretase (for example, SEQ ID NO:1). More preferably, aprotein that is structurally homologous to human beta secretase includesat least one contiguous stretch of at least 50 amino acids that sharesat least 80% amino acid sequence identity with the analogous portion ofthe native or recombinant human beta secretase (for example, SEQ IDNO:1). Methods for generating structural information about thestructurally homologous molecule or molecular complex are well-known andinclude, for example, molecular replacement techniques.

Therefore, in another embodiment this invention provides a method ofutilizing molecular replacement to obtain structural information about amolecule or molecular complex whose structure is unknown including thesteps of:

(a) crystallizing the molecule or molecular complex of unknownstructure;

(b) generating an x-ray diffraction pattern from said crystallizedmolecule or molecular complex; and

(c) applying at least a portion of the structure to the x-raydiffraction pattern to generate a three-dimensional electron density mapof the molecule or molecular complex whose structure is unknown.

By using molecular replacement, all or part of the structure coordinatesof human beta secretase or the human beta secretase/ligand complex asprovided by this invention can be used to determine the structure of acrystallized molecule or molecular complex whose structure is unknownmore quickly and efficiently than attempting to determine suchinformation ab initio.

Molecular replacement provides an accurate estimation of the phases foran unknown structure. Phases are a factor in equations used to solvecrystal structures that cannot be determined directly. Obtainingaccurate values for the phases, by methods other than molecularreplacement, is a time-consuming process that involves iterative cyclesof approximations and refinements and greatly hinders the solution ofcrystal structures. However, when the crystal structure of a proteincontaining at least a structurally homologous portion has been solved,the phases from the known structure provide a satisfactory estimate ofthe phases for the unknown structure.

Thus, this method involves generating a preliminary model of a moleculeor molecular complex whose structure coordinates are unknown, byorienting and positioning the relevant portion of human beta secretaseor the human beta secretase/inhibitor complex within the unit cell ofthe crystal of the unknown molecule or molecular complex so as best toaccount for the observed x-ray diffraction pattern of the crystal of themolecule or molecular complex whose structure is unknown. Phases canthen be calculated from this model and combined with the observed x-raydiffraction pattern amplitudes to generate an electron density map ofthe structure whose coordinates are unknown. This, in turn, can besubjected to any well-known model building and structure refinementtechniques to provide a final, accurate structure of the unknowncrystallized molecule or molecular complex (Lattman, “Use of theRotation and Translation Functions,” in Meth. Enzymol. 115, pp. 55-77(1985); M. G. Rossman, ed., “The Molecular Replacement Method,” Int.Sci. Rev. Ser. No. 13, Gordon & Breach, New York (1972)).

Structural information about a portion of any crystallized molecule ormolecular complex that is sufficiently structurally homologous to aportion of human beta secretase can be resolved by this method. Inaddition to a molecule that shares one or more structural features withhuman beta secretase as described above, a molecule that has similarbioactivity, such as the same catalytic activity, substrate specificityor ligand binding activity as human beta secretase, may also besufficiently structurally homologous to human beta secretase to permituse of the structure coordinates of human beta secretase to solve itscrystal structure.

In a preferred embodiment, the method of molecular replacement isutilized to obtain structural information about a molecule or molecularcomplex, wherein the molecule or molecular complex includes at least onehuman beta secretase subunit or homolog. A “subunit” of human betasecretase is an human beta secretase molecule that has been truncated atthe N-terminus or the C-terminus, or both. In the context of the presentinvention, a “homolog” of human beta secretase is a protein thatcontains one or more amino acid substitutions, deletions, additions, orrearrangements with respect to the amino acid sequence of human betasecretase (SEQ ID NO:1), but that, when folded into its nativeconformation, exhibits or is reasonably expected to exhibit at least aportion of the tertiary (three-dimensional) structure of human betasecretase. For example, structurally homologous molecules can containdeletions or additions of one or more contiguous or noncontiguous aminoacids, such as a loop or a domain. Structurally homologous moleculesalso include “modified” human beta secretase molecules that have beenchemically or enzymatically derivatized at one or more constituent aminoacid, including side chain modifications, backbone modifications, and N-and C-terminal modifications including acetylation, hydroxylation,methylation, amidation, and the attachment of carbohydrate or lipidmoieties, cofactors, and the like.

A heavy atom derivative of human beta secretase is also included as anhuman beta secretase homolog. The term “heavy atom derivative” refers toderivatives of human beta secretase produced by chemically modifying acrystal of human beta secretase. In practice, a crystal is soaked in asolution containing heavy metal atom salts, or organometallic compounds,e.g., lead chloride, gold thiomalate, thiomersal or uranyl acetate,which can diffuse through the crystal and bind to the surface of theprotein. The location(s) of the bound heavy metal atom(s) can bedetermined by x-ray diffraction analysis of the soaked crystal. Thisinformation, in turn, is used to generate the phase information used toconstruct three-dimensional structure of the protein (Blundell et al.,Protein Crystallography, Academic Press (1976)).

Because human beta secretase can crystallize in more than one crystalform, the structure coordinates of human beta secretase as provided bythis invention are particularly useful in solving the structure of othercrystal forms of human beta secretase or human beta secretase complexes.

The structure coordinates of human beta secretase as provided by thisinvention are particularly useful in solving the structure of human betasecretase mutants. Mutants may be prepared, for example, by expressionof human beta secretase cDNA previously altered in its coding sequenceby oligonucleotide-directed mutagenesis. Mutants may also be generatedby site-specific incorporation of unnatural amino acids into betasecretase proteins using the general biosynthetic method of Noren etal., Science 244:182-88 (1989). In this method, the codon encoding theamino acid of interest in wild-type human beta secretase is replaced bya “blank” nonsense codon, TAG, using oligonucleotide-directedmutagenesis. A suppressor tRNA directed against this codon is thenchemically aminoacylated in vitro with the desired unnatural amino acid.The aminoacylated tRNA is then added to an in vitro translation systemto yield a mutant human beta secretase with the site-specificincorporated unnatural amino acid.

Selenocysteine or selenomethionine may be incorporated into wild-type ormutant human beta secretase by expression of human betasecretase-encoding cDNAs in auxotrophic E. coli strains (Hendrickson etal., EMBO J. 9:1665-72 (1990)). In this method, the wild-type ormutagenized human beta secretase cDNA may be expressed in a hostorganism on a growth medium depleted of either natural cysteine ormethionine (or both) but enriched in selenocysteine or selenomethionine(or both). Alternatively, selenomethionine analogues may be prepared bydown regulation methionine biosynthesis. (Benson et al., Nat. Struct.Biol. 2:644-53 (1995); Van Duyne et al., J. Mol. Biol. 229:105-24(1993)).

The structure coordinates of human beta secretase are also particularlyuseful to solve the structure of crystals of human beta secretase, humanbeta secretase mutants or human beta secretase homologs co-complexedwith a variety of chemical entities. This approach enables thedetermination of the optimal sites for interaction between chemicalentities, including candidate human beta secretase inhibitors and humanbeta secretase. Potential sites for modification within the variousbinding site of the molecule can also be identified. This informationprovides an additional tool for determining the most efficient bindinginteractions, for example, increased hydrophobic interactions, betweenhuman beta secretase and a chemical entity. For example, high resolutionx-ray diffraction data collected from crystals exposed to differenttypes of solvent allows the determination of where each type of solventmolecule resides. Small molecules that bind tightly to those sites canthen be designed and synthesized and tested for their human betasecretase inhibition activity.

All of the complexes referred to above may be studied using well-knownx-ray diffraction techniques and may be refined versus 1.5-3.5 Åresolution x-ray data to an R value of about 0.30 or less using computersoftware, such as X-PLOR (Yale University, 81992, distributed byMolecular Simulations, Inc.; see, e.g., Blundell & Johnson, supra; Meth.Enzymol., Vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press(1985)). This information may thus be used to optimize known human betasecretase inhibitors, and more importantly, to design new human betasecretase inhibitors.

The invention also includes the unique three-dimensional configurationdefined by a set of points defined by the structure coordinates for amolecule or molecular complex structurally homologous to human betasecretase as determined using the method of the present invention,structurally equivalent configurations, and magnetic storage mediaincluding such set of structure coordinates.

Further, the invention includes structurally homologous molecules asidentified using the method of the invention.

Homology Modeling

Using homology modeling, a computer model of an human beta secretasehomolog can be built or refined without crystallizing the homolog.First, a preliminary model of the human beta secretase homolog iscreated by sequence alignment with human beta secretase, secondarystructure prediction, the screening of structural libraries, or anycombination of those techniques. Computational software may be used tocarry out the sequence alignments and the secondary structurepredictions. Structural incoherences, e.g., structural fragments aroundinsertions and deletions, can be modeled by screening a structurallibrary for peptides of the desired length and with a suitableconformation. For prediction of the side chain conformation, a sidechain rotamer library may be employed. If the human beta secretasehomolog has been crystallized, the final homology model can be used tosolve the crystal structure of the homolog by molecular replacement, asdescribed above. Next, the preliminary model is subjected to energyminimization to yield an energy minimized model. The energy minimizedmodel may contain regions where stereochemistry restraints are violated,in which case such regions are remodeled to obtain a final homologymodel. The homology model is positioned according to the results ofmolecular replacement, and subjected to further refinement includingmolecular dynamics calculations.

Rational Drug Design

Computational techniques can be used to screen, identify, select and/ordesign chemical entities capable of associating with human betasecretase or structurally homologous molecules. Knowledge of thestructure coordinates for human beta secretase permits the design and/oridentification of synthetic compounds and/or other molecules which havea shape complementary to the conformation of the human beta secretasebinding site. In particular, computational techniques can be used toidentify or design chemical entities, such as inhibitors, agonists andantagonists, that associate with an human beta secretase inhibitorbinding pocket or an beta secretase-like inhibitor binding pocket.Inhibitors may bind to or interfere with all or a portion of an activesite of human beta secretase, and can be competitive, non-competitive,or uncompetitive inhibitors; or interfere with dimerization by bindingat the interface between the two monomers. Once identified and screenedfor biological activity, these inhibitors/agonists/antagonists may beused therapeutically or prophylactically to block human beta secretaseactivity and, thus, prevent the onset and/or further progression ofAlzheimer's disease. Structure-activity data for analogues of ligandsthat bind to or interfere with human beta secretase or betasecretase-like inhibitor binding pockets can also be obtainedcomputationally.

The term “chemical entity,” as used herein, refers to chemicalcompounds, complexes of two or more chemical compounds, and fragments ofsuch compounds or complexes. Chemical entities that are determined toassociate with human beta secretase are potential drug candidates.

Data stored in a machine-readable storage medium that is capable ofdisplaying a graphical three-dimensional representation of the structureof human beta secretase or a structurally homologous molecule, asidentified herein, or portions thereof may thus be advantageously usedfor drug discovery. The structure coordinates of the chemical entity areused to generate a three-dimensional image that can be computationallyfit to the three-dimensional image of human beta secretase or astructurally homologous molecule. The three-dimensional molecularstructure encoded by the data in the data storage medium can then becomputationally evaluated for its ability to associate with chemicalentities. When the molecular structures encoded by the data is displayedin a graphical three-dimensional representation on a computer screen,the protein structure can also be visually inspected for potentialassociation with chemical entities.

One embodiment of the method of drug design involves evaluating thepotential association of a known chemical entity with human betasecretase or a structurally homologous molecule, particularly with anhuman beta secretase inhibitor binding pocket or beta secretase-likeinhibitor binding pocket. The method of drug design thus includescomputationally evaluating the potential of a selected chemical entityto associate with any of the molecules or molecular complexes set forthabove. This method includes the steps of: (a) employing computationalmeans to perform a fitting operation between the selected chemicalentity and a inhibitor binding pocket or a pocket nearby the inhibitorbinding pocket of the molecule or molecular complex; and (b) analyzingthe results of said fitting operation to quantify the associationbetween the chemical entity and the inhibitor binding pocket.

In another embodiment, the method of drug design involvescomputer-assisted design of chemical entities that associate with humanbeta secretase, its homologs, or portions thereof. Chemical entities canbe designed in a step-wise fashion, one fragment at a time, or may bedesigned as a whole or “de novo.”

To be a viable drug candidate, the chemical entity identified ordesigned according to the method must be capable of structurallyassociating with at least part of an human beta secretase or betasecretase-like inhibitor binding pockets, and must be able, stericallyand energetically, to assume a conformation that allows it to associatewith the human beta secretase or beta secretase-like inhibitor bindingpocket. Non-covalent molecular interactions important in thisassociation include hydrogen bonding, van der Waals interactions,hydrophobic interactions, and electrostatic interactions. Conformationalconsiderations include the overall three-dimensional structure andorientation of the chemical entity in relation to the inhibitor bindingpocket, and the spacing between various functional groups of an entitythat directly interact with the beta secretase-like inhibitor bindingpocket or homologs thereof.

Optionally, the potential binding of a chemical entity to an human betasecretase or beta secretase-like inhibitor binding pocket is analyzedusing computer modeling techniques prior to the actual synthesis andtesting of the chemical entity. If these computational experimentssuggest insufficient interaction and association between it and thehuman beta secretase or beta secretase-like inhibitor binding pocket,testing of the entity is obviated. However, if computer modelingindicates a strong interaction, the molecule may then be synthesized andtested for its ability to bind to or interfere with an human betasecretase or beta secretase-like inhibitor binding pocket. Bindingassays to determine if a compound actually interferes with human betasecretase can also be performed and are well known in the art. Bindingassays may employ kinetic or thermodynamic methodology using a widevariety of techniques including, but not limited to, microcalorimetry,circular dichroism, capillary zone electrophoresis, nuclear magneticresonance spectroscopy, fluorescence spectroscopy, and combinationsthereof.

One skilled in the art may use one of several methods to screen chemicalentities or fragments for their ability to associate with an human betasecretase or beta secretase-like inhibitor binding pocket. This processmay begin by visual inspection of, for example, an human beta secretaseor beta secretase-like inhibitor binding pocket on the computer screenbased on the human beta secretase structure coordinates or othercoordinates which define a similar shape generated from themachine-readable storage medium. Selected fragments or chemical entitiesmay then be positioned in a variety of orientations, or docked, withinthe inhibitor binding pocket. Docking may be accomplished using softwaresuch as QUANTA and SYBYL, followed by energy minimization and moleculardynamics with standard molecular mechanics forcefields, such as CHARMMand AMBER.

Specialized computer programs may also assist in the process ofselecting fragments or chemical entities. Examples include GRID(Goodford, J. Med. Chem. 28:849-57 (1985); available from OxfordUniversity, Oxford, UK); MCSS (Miranker et al., Proteins: Struct. Funct.Gen. 11:29-34 (1991); available from Molecular Simulations, San Diego,Calif.); AUTODOCK (Goodsell et al., Proteins: Struct. Funct. Genet.8:195-202 (1990); available from Scripps Research Institute, La Jolla,Calif.); and DOCK (Kuntz et al., J. Mol. Biol. 161:269-88 (1982);available from University of California, San Francisco, Calif.).

Once suitable chemical entities or fragments have been selected, theycan be assembled into a single compound or complex. Assembly may bepreceded by visual inspection of the relationship of the fragments toeach other on the three-dimensional image displayed on a computer screenin relation to the structure coordinates of human beta secretase. Thiswould be followed by manual model building using software such as QUANTAor SYBYL (Tripos Associates, St. Louis, Mo.).

Useful programs to aid one of skill in the art in connecting theindividual chemical entities or fragments include, without limitation,CAVEAT (Bartlett et al., in Molecular Recognition in Chemical andBiological Problems,” Special Publ., Royal Chem. Soc., 78:182-96 (1989);Lauri et al., J. Comput. Aided Mol. Des. 8:51-66 (1994); available fromthe University of California, Berkeley, Calif.); 3D database systemssuch as ISIS (available from MDL Information Systems, San Leandro,Calif.; reviewed in Martin, J. Med. Chem. 35:2145-54 (1992)); and HOOK(Eisen et al., Proteins: Struc., Funct., Genet. 19:199-221 (1994);available from Molecular Simulations, San Diego, Calif.).

Human beta secretase binding compounds may be designed “de novo” usingeither an empty binding site or optionally including some portion(s) ofa known inhibitor(s). There are many de novo ligand design methodsincluding, without limitation, LUDI (Bohm, J. Comp. Aid. Molec. Design.6:61-78 (1992); available from Molecular Simulations Inc., San Diego,Calif.); LEGEND (Nishibata et al., Tetrahedron, 47:8985 (1991);available from Molecular Simulations Inc., San Diego, Calif.); LeapFrog(available from Tripos Associates, St. Louis, Mo.); and SPROUT (Gilletet al., J. Comput. Aided Mol. Design. 7:127-53 (1993); available fromthe University of Leeds, UK).

Once a compound has been designed or selected by the above methods, theefficiency with which that entity may bind to or interfere with an humanbeta secretase or beta secretase-like inhibitor binding pocket may betested and optimized by computational evaluation. For example, aneffective human beta secretase or beta secretase-like inhibitor bindingpocket inhibitor must preferably demonstrate a relatively smalldifference in energy between its bound and free states (i.e., a smalldeformation energy of binding). Thus, the most efficient human betasecretase or beta secretase-like inhibitor binding pocket inhibitorsshould preferably be designed with a deformation energy of binding ofnot greater than about 10 kcal/mole; more preferably, not greater than 7kcal/mole. human beta secretase or beta secretase-like inhibitor bindingpocket inhibitors may interact with the inhibitor binding pocket in morethan one conformation that is similar in overall binding energy. Inthose cases, the deformation energy of binding is taken to be thedifference between the energy of the free entity and the average energyof the conformations observed when the inhibitor binds to the protein.

An entity designed or selected as binding to or interfering with anhuman beta secretase or beta secretase-like inhibitor binding pocket maybe further computationally optimized so that in its bound state it wouldpreferably lack repulsive electrostatic interaction with the targetenzyme and with the surrounding water molecules. Such non-complementaryelectrostatic interactions include repulsive charge-charge,dipole-dipole, and charge-dipole interactions.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interactions. Examples of programsdesigned for such uses include: Gaussian 94, revision C (M. J. Frisch,Gaussian, Inc., Pittsburgh, Pa. 81995); AMBER, version 4.1 (P. A.Kollman, University of California at San Francisco, 81995);QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif. 81995);Insight II/Discover (Molecular Simulations, Inc., San Diego, Calif.81995); DelPhi (Molecular Simulations, Inc., San Diego, Calif. 81995);and AMSOL (Quantum Chemistry Program Exchange, Indiana University).These programs may be implemented, for instance, using a SiliconGraphics workstation such as an Indigo² with “IMPACT” graphics. Otherhardware systems and software packages will be known to those skilled inthe art.

Another approach encompassed by this invention is the computationalscreening of small molecule databases for chemical entities or compoundsthat can bind in whole, or in part, to a human beta secretase or betasecretase-like inhibitor binding pocket. In this screening, the qualityof fit of such entities to the binding site may be judged either byshape complementarity or by estimated interaction energy (E. C. Meng etal., J. Comp. Chem. 13:505-24 (1992)).

This invention also enables the development of chemical entities thatcan isomerize to short-lived reaction intermediates in the chemicalreaction of a substrate or other compound that interferes with or withhuman beta secretase. Time-dependent analysis of structural changes inhuman beta secretase during its interaction with other molecules iscarried out. The reaction intermediates of human beta secretase can alsobe deduced from the reaction product in co-complex with human betasecretase. Such information is useful to design improved analogues ofknown human beta secretase inhibitors or to design novel classes ofinhibitors based on the reaction intermediates of the human betasecretase and inhibitor co-complex. This provides a novel route fordesigning human beta secretase inhibitors with both high specificity andstability.

Yet another approach to rational drug design involves probing the humanbeta secretase crystal of the invention with molecules including avariety of different functional groups to determine optimal sites forinteraction between candidate human beta secretase inhibitors and theprotein. For example, high resolution x-ray diffraction data collectedfrom crystals soaked in or co-crystallized with other molecules allowsthe determination of where each type of solvent molecule sticks.Molecules that bind tightly to those sites can then be further modifiedand synthesized and tested for their beta secretase inhibitor activity(Travis, Science 262:1374 (1993)).

In a related approach, iterative drug design is used to identifyinhibitors of human beta secretase. Iterative drug design is a methodfor optimizing associations between a protein and a compound bydetermining and evaluating the three-dimensional structures ofsuccessive sets of protein/compound complexes. In iterative drug design,crystals of a series of protein/compound complexes are obtained and thenthe three-dimensional structures of each complex is solved. Such anapproach provides insight into the association between the proteins andcompounds of each complex. This is accomplished by selecting compoundswith inhibitory activity, obtaining crystals of this newprotein/compound complex, solving the three dimensional structure of thecomplex, and comparing the associations between the new protein/compoundcomplex and previously solved protein/compound complexes. By observinghow changes in the compound affected the protein/compound associations,these associations may be optimized.

A compound that is identified or designed as a result of any of thesemethods can be obtained (or synthesized) and tested for its biologicalactivity, e.g., inhibition of beta secretase activity.

Pharmaceutical Compositions (Inhibitors)

Pharmaceutical compositions of this invention include an inhibitor ofhuman beta secretase activity identified according to the invention, ora pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle. The term “pharmaceuticallyacceptable carrier” refers to a carrier(s) that is “acceptable” in thesense of being compatible with the other ingredients of a compositionand not deleterious to the recipient thereof. Optionally, the pH of theformulation is adjusted with pharmaceutically acceptable acids, bases,or buffers to enhance the stability of the formulated compound or itsdelivery form.

Methods of making and using such pharmaceutical compositions are alsoincluded in the invention. The pharmaceutical compositions of theinvention can be administered orally, parenterally, by inhalation spray,topically, rectally, nasally, buccally, vaginally, or via an implantedreservoir. Oral administration or administration by injection ispreferred. The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intra-articular,intrasynovial, intrasternal, intrathecal, intralesional, andintracranial injection or infusion techniques.

Dosage levels of between about 0.01 and about 100 mg/kg body weight perday, preferably between about 0.5 and about 75 mg/kg body weight per dayof the human beta secretase inhibitory compounds described herein areuseful for the prevention and treatment of human beta secretase mediateddisease. Typically, the pharmaceutical compositions of this inventionwill be administered from about 1 to about 5 times per day oralternatively, as a continuous infusion. Such administration can be usedas a chronic or acute therapy. The amount of active ingredient that maybe combined with the carrier materials to produce a single dosage formwill vary depending upon the host treated and the particular mode ofadministration. A typical preparation will contain from about 5% toabout 95% active compound (w/w). Preferably, such preparations containfrom about 20% to about 80% active compound.

In order that this invention be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

EXAMPLES Crystallization and Structure Determination of Human BetaSecretase

A. Expression, Purification, and Crystallization

Expression and Purification of Beta Secretase from HEK 293 Cells.

The expression plasmid=pcDNA3.1/myc/his (neomycin) (Invitrogen) containsbeta secretase extending from Met [−21] to Ser [432] with a myc tagfollowed by a hexahistidine tag [EQKLISEEDLMHTEHHHHHH*] (SEQ ID NO:2) atthe C-terminus. Following transfection in HEK293 cells, stable cellswere selected using 0.8 mg/ml G418. A stable clone of transfected HEK293cells that secretes human beta-secretase was expanded in static,monolayer cell culture. Confluent cultures were detached by shaking anda plurality of plastic, 225 cm² T-flasks were inoculated with asuspension of 1−5×106 cells in 100 ml of High-Glucose Dulbecco'sModified Eagle medium that was supplemented with 5% fetal bovine serumand 500 micrograms/ml G418. These cell cultures were incubated in ahumidified 37° C. incubator gassed with 95% air and 5% CO₂. Once thecells reached confluence the growth medium in each flask was removed andreplaced with 100 ml fresh medium. The conditioned, culture mediumsupernatant was harvested aseptically and replaced by fresh medium every48-72 hours. The harvested medium was pooled, centrifuged at 1000×g toremove cell debris, and was stored in plastic bottles at 4° C. Cellmonolayers were maintained in semi-continuous culture for several weeksuntil the cells either began to die or to detach from the cultureflasks. The cells were then resuspended and used to inoculate a freshset of production flasks.

For purification, the medium was concentrated approximately 10-foldusing a tangential flow concentrator equipped with a 30,000 molecularweight cutoff cartridge. Solid ammonium sulfate was then slowly addedwith stirring to the concentrate at 4° C. to a final value of 40%saturation (242 g/L). After stirring at 4° C. for 30 minutes, thesuspension was clarified by centrifugation (16,000×g, 60 minutes) andthe supernatant taken for further analysis. The 40% ammonium sulfatesupernatant was adjusted to 80% saturation by slow addition of solidammonium sulfate with stirring at 4° C. (281 g/L). After stirring for 30minutes at 4° C., the insoluble material was collected by centrifugationas indicated above and the 40-80% ammonium sulfate pellet taken forfurther analysis.

The 40-80% ammonium sulfate pellet was dissolved in 25 mM Tris-HCl (pH8.5)/0.5 M NaCl/10 mM imidazole ( 1/10 the original volume) and appliedto a 12.5 ml column containing Ni⁺-NTA Last Flow resin previouslyequilibrated in the same buffer. Following sample application, thecolumn was washed with 10 column volumes of loading buffer and theneluted with 25 mM Tris-HCl (pH 8.5)/0.5 M NaCl/50 mM imidazole. Thematerial eluting in 50 mM imidazole was pooled, concentratedapproximately 10-fold using a YM 30 membrane (30,000 MWCO), and thendialyzed against 10 mM HEPES-Na (pH 8.0) using 50,000 molecular weightcutoff tubing. For affinity purification, the synthetic peptideSer-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Arg-Gly-Gly-Cys (where Sta=statine,PNU-292593E) (SEQ ID NO:3) was synthesized and coupled to sulfolinkresin (Pierce Chemical Company, Rockford, Ill.) as recommended by themanufacture. The dialyzed material from above was adjusted to 0.1 MNaOAc (pH 4.5) by addition of 1/10 volume of 1.0 M NaOAc (pH 4.5) andimmediately applied to the PNU-292593E/sulfolink column (6 ml containing1.0 mg of the synthetic peptide shown in FIG. 2/ml of resin) that hadbeen previously equilibrated in 25 mM NaOAc (pH 4.5). Following sampleapplication, the column was washed with 10 column volumes of 25 mM NaOAc(pH 4.5) and then eluted with 50 mM NaBO₃ (pH 8.5). N-terminal sequenceanalysis of the affinity purified material revealed an equimolar mixtureof pro- and processed human β-secretase beginning at Thr¹ and Glu²⁵,respectively. The final protein concentration was determined by aminoacid analysis assuming a 60 kDa glycoprotein.

Production of Recombinant Human β-Secretase in Insect sf9 Cells andCHO-K1 Cells. The coding sequence was engineered to delete the terminaltransmembrane and cytoplasmic domain and introduce a C-terminalhexahistidine tag using the polymerase chain reaction. The 5′ senseoligonucleotide primer [CGCTTTGGATCCGTGGACAACCTGAGGGGCAA] (SEQ ID NO:4)was designed to incorporate a BamHI site for ease in subcloning andKozak consensus sequence around the initiator methionine for optimaltranslation initiation. The 3′ antisense primer[CGCTTTGGTACCCTATGACTCATCTGTCTGTGGAATGTTG] (SEQ ID NO:5) incorporated ahexahistidine tag and translation termination codon just upstream of thepredicted transmembrane domain (Ser⁴³²) and a NotI restriction site forcloning. The PCR was performed on the plasmid templatepcDNA3.1hygroAsp2R for 15 cycles [94° C., 30 sec., 65° C., 30 sec., 72°C., 30 sec] using Pwo I polymerase (Roche Biochemicals, Indianapolis,Ind.) as outlined by the manufacturer. The PCR product was digested tocompletion with BamHI and NotI and ligated into the BamHI and NotI sitesof the Baculovirus transfer vector pVL1393 (PharMingen, San Diego,Calif.). A portion of the ligation was used to transform competent E.coli DH5a cells and recombinant clones were selected on ampicillin.Individual clones containing the proper cDNA inserts were identified byPCR. Plasmid DNA from clone (pVL1393/Hu_Asp-2LΔTM(His)₆) was prepared byalkaline lysis and banding in CsCl. The integrity of the insert wasconfirmed by complete DNA sequencing. For CHO-K¹ cell expression,plasmid pVL1393/Hu_Asp-2LΔTM(His)₆ was digested with BamHI and NotI andthe resulting fragment subcloned into the mammalian expression vectorpcDNA3.1 (hygro) as described above to yield pcDNA3.1(hygro)/Hu_Asp-2LΔTM(His)₆).

For expression, CHO-K1 cells (50% confluent) were transfected withcationic liposome/pcDNA3.1 (hygro)/Hu_Asp-2LΔTM(His)₆ complexes in α-MEMmedium containing 10% FBS overnight. Selection was performed in the samemedium containing 0.5 mg/L hygromycin B for seven days and survivingcells were cloned by limiting dilution. Eight cell lines were screenedfor soluble β-secretase by Western blot analysis using a polyclonalrabbit antiserum specific for human β-secretase (UP-191). Conditionedmedium from each clonal cell line was concentrated by Ni⁺-NTAchromatography and the histidine-tagged polypeptide eluted with buffercontaining 50 mM imidazole. Aliquots of the latter fraction weredisplayed on a PVDF membrane and recombinant soluble human P-secretasewas visualized using UP-191 antiserum and alkaline phosphataseconjugated goat antirabbit second antibody. Based on these results,clone #4 showed the highest expression level and was used for allsubsequent experiments.

For Baculovirus expression (BVES), recombinant virus containing thecoding sequence of soluble β-secretase was isolated followingrecombination of the plasmid transfer vector pVL1393/Hu_Asp-2LΔTM(His)₆in sf9 cells using standard methods. Individual virus isolates were usedto infect sf9 cells and expression of the desired polypeptide wasquantified by Western blot analysis of the conditioned medium collected69 hr post infection as described above. The recombinant virus directingthe synthesis of the highest level of β-secretase was scaled-up forprotein production.

For production of soluble β-secretase in insect cells, sf9 cells wereinfected with recombinant Baculovirus at a multiplicity of infection of1.0 in serum free medium and conditioned medium harvested 69 hourspost-infection. For CHO cells, the production cell line expressingsecreted soluble human β-secretase was expanded in either roller bottlesor in a packed-bed bioreactor in medium containing 0.5 fetal bovineserum. Conditioned medium was collected and stored at 4° C. untilprocessing.

Purification of Recombinant Human β-Secretase from BVES or CHO-K1 Cells.For purification from either source, the medium was concentratedapproximately 10-fold using a tangential flow concentrator equip with a30,000 molecular weight cutoff cartridge. Solid ammonium sulfate wasthen slowly added with stirring to the concentrate at 4° C. to a finalvalue of 40% saturation (242 g/L). After stirring at 4° C. for 30minutes, the suspension was clarified by centrifugation (16,000×g, 60minutes) and the supernatant taken for further analysis. The 40%ammonium sulfate supernatant was adjusted to 80% saturation by slowaddition of solid ammonium sulfate with stirring at 4° C. (281 g/L).After stirring for 30 minutes at 4° C., the insoluble material wascollected by centrifugation as indicated above and the 40-80% ammoniumsulfate pellet taken for further analysis.

The 40-80% ammonium sulfate pellet was dissolved in 25 mM Tris-HCl (pH8.5)/0.5 M NaCl/10 mM imidazole ( 1/10 the original volume) and appliedto a 12.5 ml column containing Ni⁺-NTA Fast Flow resin previouslyequilibrated in the same buffer. Following sample application, thecolumn was washed with 10 column volumes of loading buffer and theneluted with 25 mM Tris-HCl (pH 8.5)/0.5 M NaCl/50 mM imidazole. Thematerial eluting in 50 mM imidazole was pooled, concentratedapproximately 10-fold using a YM 30 membrane (30,000 MWCO), and thendialyzed against 10 mM HEPES-Na (pH 8.0) using 50,000 molecular weightcutoff tubing. For affinity purification, the synthetic peptideSer-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Arg-Gly-Gly-Cys (where Sta=statine,PNU-292593E) (SEQ ID NO:3) was synthesized and coupled to sulfolinkresin (Pierce Chemical Company, Rockford, Il) as recommended by themanufacture. The dialyzed material from above was adjusted to 0.1 MNaOAc (pH 4.5) by addition of 1/10 volume of 1.0 M NaOAc (pH 4.5) andimmediately applied to the PNU-292593E/sulfolink column (6 ml containing1.0 mg of the synthetic peptide shown in FIG. 2/ml of resin) that hadbeen previously equilibrated in 25 mM NaOAc (pH 4.5). Following sampleapplication, the column was washed with 10 column volumes of 25 mM NaOAc(pH 4.5) and then eluted with 50 mM sodium borate (pH 8.5). N-terminalsequence analysis of the affinity purified material revealed anequimolar mixture of pro- and processed human β-secretase beginning atThr¹ and Glu²⁵, respectively. The final protein concentration wasdetermined by amino acid analysis assuming a 52 kDa glycoprotein forinsect cells and a 60 kDa glycoprotein for CHO cells, respectively.

Protein Crystallization. Based on observations of the initial screeningeffort, fresh protein derived from CHO cells was concentrated to 42mg/ml and mixed with the inhibitor shown in FIG. 1 so that the finalconcentration of the mix was 40 mg/ml beta secretase, and 2 mM of theinhibitor shown in FIG. 1, in 20 mM Hepes pH 7.8, 10% DMSO. Thispreparation was screened with Hampton Screen 1 (Hampton Research, LagunaNigel, Calif.) and Wizard Screen 1 (Emerald Biostructures, BainbridgeIsland, Wash.) at room temperature (20° C.). 500-μL well volumes wereused. A 1:1 ratio of protein-compound mix to the well solution was usedin a hanging drop format to complete the screen. After 10 days, but lessthan 18 days later crystals were observed in Wizard Screen 1, ConditionNo. 45 (20% PEG 3000, 0.1M NaOAc⁻ pH 4.5). Optimization and seedingefforts around this condition provided crystals that grew in 17-20% PEG3000, 0.1M Na Acetate pH 4.5. Seeding was done utilizing a cat whiskerwhich was touched to a drop containing microcrystals and stepwisediluted by streaking through one row of the optimization tray.Cross-seeding efforts provided crystals of HEK 293 cell derived protein(38 mg/ml in 20 mM Hepes pH 7.8, 50 mM NaCl, 10% DMSO, 2 mM of theinhibitor shown in FIG. 1) from CHO cell derived seed stock.Macroseeding by moving small crystals with a loop from the target dropto a fresh drop containing 17-20% PEG3000, 0.1M Na Acetate pH 4.5 alsoresulted in crystals that doubled or more in size, usually with a showerof microcrystals also. Crystals were obtained in hanging drop or sittingdrop methods by seeding. Crystals obtained from streak seeding attemptswere frozen in a cryoprotectant solution based on the mother liquor plus20% Ethylene Glycol. The crystals were then soaked incrementally through5% intervals of the cryoprotectant in 3 to 5-minute intervals. Crystalshave also been grown in the presence of 10% glycerol or 10% ethyleneglycol to facilitate stabilization into cryogenic solutions. In thesecases, the crystals were soaked incrementally through 5% intervals ofthe cryoprotectant in 3 to 5-minute intervals. The use of other cryoagents would be apparent to one of skill in the art.

Fresh Asp2 protein derived from Baculovirus cells (BVES) wasconcentrated to 18.6 mg/mL and mixed with the inhibitor illustrated inFIG. 1, so that the final concentration of the mix was 18 mg/mL betasecretase and 2 mM of the inhibitor illustrated in FIG. 1 in 20 mM HepespH 7.8 with 5% DMSO. Observations of how CHO derived Asp2 crystallizedled to the following crystallization effort for BVES Asp2. A PEG rangefrom 17-22% PEG 3000, 0.1 M sodium acetate pH 4.5, with none, 10% DMSO,or 10% glycerol was used in 1000 μL wells. Sample drops were streakseeded at set up, with 10⁻¹ seed dilution of CHO seed stocks asdescribed above. A crystal measuring 1.0×0.1×0.1 mm3 was obtained in 17%PEG 3000, 0.1M sodium acetate pH 4.5 with 10% DMSO, and 3 days post setup. Crystals were incrementally soaked into a cryoprotectant solutioncontaining a final concentration of 20% DMSO in 5 minute intervals.

B. X-Ray Diffraction Characterization for Beta Secretase Expressed inCHO Cells

All data collection was carried out at the Advanced Photon Source(Argonne, Ill.) beamline 17-ID. The crystals diffracted to 3.2 Å usingsynchrotron radiation. Crystals were of the space group P3₂21 with cellconstants a=112±20 Å, b=112±20 Å, c=110±20 Å, α=β=90°, γ=120°. TheMatthews coefficient for these crystals assuming that there is onemolecules in the asymmetric unit is 3.5 Å/Da with 65% solvent. Thestructure determination (see below) revealed the presence of electrondensity in the active site appropriate for the inhibitor shown in FIG.1.

C. Molecular Replacement for Beta Secretase Expressed in CHO Cells

A molecular replacement solution was determined using AMORE (Navaza,Acta Cryst., D50:157-63 (1994); Collaborative Computational Project N4′,Acta Cryst. D50:760-3 (1994)) by utilizing a previously published modelof human beta secretase, 1FKN, (Hong et al., Science 290: 150-53 (2000))made available from the Protein Data Bank (world wide web at rcsb.org).Using the 1FKN model, the initial rotation solution gave a single strongpeak of 9.7σ with the next strongest peak appearing at 4σ. The finaldetermination of the space group (P321, P3₁21, or P3₂21) was determinedexperimentally by testing translation searches in each space group. Atranslation search in the correct space group, P3₂21, resulted in acorrelation coefficient of 55.1 with an R-factor of 39.9% to 4 Åresolution.

TABLE 4 Data collection statistics for structure of Human Beta Secretase(data collected at λ 1.0000 Å at APS, 17-ID) Resolution Range R_(sym)  20-6.81 0.048 6.81-5.44 0.095 5.44-4.77 0.112 4.77-4.33 0.1134.33-4.03 0.181 4.03-3.79 0.224 3.79-3.60 0.248 3.60-3.45 0.2883.45-3.31 0.321 3.31-3.20 0.324 All reflections 0.098D. Model Building and Refinement for Beta Secretase Expressed in CHOCells

Further rigid body refinement of the model in CNX (MolecularSimulations, Inc) followed by minimization and group b-factor refinementgave an R-factor of 35.1% and a Free R-factor of 37.7% to 3.2 Å. Duringeach cycle of refinement a bulk solvent correction was incorporated(Jiang et al., J. Mol. Biol. 243:100-15 (1994)). Progress of therefinement was monitored by a decrease in both the R-factor and FreeR-factor.

At this point, inspection of the electron density map within the activesite revealed electron density that was unaccounted for by the proteinmodel and consistent with the shape of the inhibitor shown in FIG. 1Bthat was present in the crystallization conditions. Model building wasdone using the program CHAIN (Sack, Journal of Molecular Graphics6:224-25 (1988)) and LORE (Finzel, Meth. Enzymol. 277:230-42 (1997)).Modest rebuilding of the model into the 3.2 Å low resolution mapafforded the opportunity for further cycles of refinement givingimprovement of the R-factor to 31.6% and a Free R-factor of 35.7%.Finally, the inhibitor was included in the refinement to give thecurrent R-factor of 29.9% and a Free R-factor of 34.9%.

Inspection of the electron density throughout the molecule indicatesthat all three disulfide bonds are intact (Cys155-Cys359,Cys217-Cys382,and Cys269-Cys 319). In addition, the electron density near Asn92,Ans162, and Asn293, indicates the presence of glycosylation. Only theglycosylation at Asn111 is disordered enough not to be visible in theelectron density map. Residues 158-170 and 311 to 317 were disordered inthe electron density and therefore have been omitted from the model.

TABLE 5 Refinement Statistics for structure of Human Beta Secretase No.of R-factor Free R-factor reflections 20-3.2 Å 0.2991 0.3483 9883 F ≧ 2σBonds (Å) Angles(°) r.m.s deviation from ideal geometry 0.012 1.7 Numberof atoms Average B-factor Protein 2880 65.24 Waters 41 74.89 Total 292165.38E. X-ray Diffraction Characterization for Baculovirus Produced Humanbeta Secretase Crystals.

All data collection was carried out at the Advanced Photon Source(Argonne, Ill.) at beamline 17-ID. The crystals diffracted to 2.7 Åusing synchrotron radiation. Crystals were of the space group P3₂21 withcell constants a=99.4±35 Å, b=99.4±35 Å, c=117±35 Å, α=β=90°, γ=120°.The Matthews coefficient for these crystals assuming that there is onemolecule in the asymmetric unit is 2.9 Å/Da with 58% solvent. Thestructure determination (see below) revealed the presence of electrondensity in the active site appropriate for the inhibitor shown in FIG.1.

F. Molecular Replacement for Baculovirus Produced Human Beta SecretaseCrystals.

All molecular replacement solution was determined using AMORE (Navaza1994; Collaborative Computational Project N4, Acta Cryst. D50:760-3 byutilizing the structure of human beta secretase produced from CHO cells.Using the CHO beta secretase model, the initial rotation solution gave astrong peak of 10.2σ with the strongest peak appearing at 8.0σ. Thespace group was P3₂21. A translation search in the correct space group,P3₂21, resulted in a correlation of 57.8 with an R-Factor of 41.5% to 4Å resolution.

TABLE 6 Data collection statistics for structure of Human Beta Secretaseexpressed in a Baculovirus expression system (data collected at λ 1.0000Å at APS, 17-ID) Resolution Range R_(sym) 99.0-5.81 0.045 5.81-4.610.076 4.61-4.03 0.093 4.03-3.66 0.117 3.66-3.40 0.139 3.40-3.19 0.1763.19-3.03 0.149 3.03-2.90 0.174 2.90-2.79 0.169 2.79-2.69 0.273 Allreflections 0.073G. Model Building and Refinement for Baculovirus Produced Human BetaSecretase Crystals.

Further rigid body refinement of the model in CNX (MolecularSimulations, Inc) followed by minimization refinement gave an R-factorof 36.4% to 2.7 Å. During each cycle of refinement a bulk solventcorrection was incorporated (Jiang et al., J. Mol. Biol. 243:100-15(1994)).

At this point, inspection of the electron density map within the activesite revealed electron density that was unaccounted for by the proteinmodel and consistent with the shape of the inhibitor illustrated in FIG.1, that was present in the crystallization conditions. Model buildingwas done using the program CHAIN (Sack, Journal of Molecular Graphics6:224-25 (1988)) and LORE (Finzel, Meth. Enzymol. 277:23042 (1997)).

The complete disclosure of all patents, patent applications includingprovisional applications, and publications, and electronically availablematerial (e.g., GenBank amino acid and nucleotide sequence submissions)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described; manyvariations will be apparent to one skilled in the art and are intendedto be included within the invention defined by the claims.

SEQUENCE LISTING FREE TEXT

-   SEQ ID NO:1 residues for recombinant human beta secretase present in    the X-ray structure

1. A crystal of beta secretase co-crystallized with a ligand, whereinthe amino acid sequence of said beta secretase is SEQ ID NO:1, saidcrystal has space group symmetry P3₂21, a unit cell defined bydimensions a, b, c, α, β and γ, wherein a is 99.4±35 Å, b is 99.4±35 Å,c is 117±35 Å, α=β=90°, γ=120°, and wherein said ligand is bound at theactive site of said beta secretase, and isN1-((2S,3R)-4-(3-iodobenzylamino)-1-(3,5-difluorophenyl)-3-hydroxybutan-2-yl)-5-methyl-N3,N3-dipropylbenzene-1,3-diamide.2. A crystal of beta secretase co-crystallized with a ligand, whereinthe amino acid sequence of said beta secretase is SEQ ID NO:1, whereinat least one methionine is replaced by selenomethionine, said crystalhas space group symmetry P3₂21, a unit cell defined by dimensions a, b,c, α, β and γ, wherein a is 99.4±35 Å, b is 99.4±35 Å, c is 117±35 Å,α=β=90°, γ=120°, and wherein said ligand is bound at the active site ofsaid beta secretase, and isN1-((2S,3R)-4-(3-iodobenzylamino)-1-(3,5-difluorophenyl)-3-hydroxybutan-2-yl)-5-methyl-N3,N3-dipropylbenzene-1,3-diamide.